Title of Invention

THE PROCESS FOR THE SYNTHESIS OF BISBENZIMIDAZOLES AND ITS DERIVATIONS

Abstract This invention relates to a process for the synthesis of bisbenzimidazoles and its derivations comprising reacting 5 chloroaniline with zinc dust and acetic anhydride to produce 5 chloroacetanilide; reacting 5 chloroacetanilide with HNO3 to produce 2-nitro-5- chloroacetanilide; boiling sodium methoxide and 2-nitro-5- chloroacetanilide under reflux for 3 hours to prepare 2-nitro-5- chloroaniline; heating 2-nitro-5-chloroaniline, methyl piperazme, anhydrous K2CO3 and Dimethyl formamide at 100-120°C produce a mixture which is cooled by pouring ice and is filtered to obtain 5-(4'- methylpiperazin-1 '-yl)-2-nitroaniline; treating 5-(4'-methylpiperazin-1 '- yl)-2-nitroaniline with Pd/C to produce 2-amino-4-(4'-methylpiperazin-l'- yl) aniline; refluxing a mixture of 2-amino-4-(4'-methylpiperazin-l'-yl) aniline and ethyl-4-amino-3-nitrobenzenecarboximidate hydrochloride in presence of ethanol/glacial acetic acid to produce 4-[5'-(4"- methylpiperazin-l"-yl) benzimidazol-2'-yl)-2-nitroaniline; treating a solution of 4-[5'-(4"-methylpiperazin-l"-yl) benzimidazol-2'-yl)-2- nitroaniline with palladium on carbon to yield 2-amino-4-[5'-(4"- Methylpiperazin- l"-yl)benzimidazol-2'-yl]aniline; heating 2-amino-4-[5'- (4"-Methylpiperazin-l"-yl) benzimidazol-2'-yl] aniline and 3-4-dimethoxy benzaldehyde using nitrobenzene as a solvent at 110-150°C to produce (DMA) i.e. 5-(4-methylpiperazin-l-yl)2-2'-3,4-dimethoxyphenyl)- 5'benzimidazolyl]benzimidazole; heating 2-amino-4-[5'-(4"- Methylpiperazin-l"-yl) benzimidazol-2'- yl] aniline and 5-Formyl-[3-methoxy-4-hydroxy benzimidazole] using nitrobenzene at 110°C to 150°C in presence of argon to produce (TBZ) i.e. 5-(4-methylpiperazine-l-yl)-2-[2'{2':-(4-hydroxy-3 methoxyphenyl)5"benzimidazolyl} -5'-benzimidazolyl] benzimidazole.
Full Text FIELD OF THE INVENTION
This invention relates to the process tor the synthesis of bisbenzimidazoles and its derivations.
BACKGROUND OF THE INVENTION
The DNA Uganda such as bisbenzimidazoles Hoechst 33342 and Hoechst 332S8, torm strong and non-covalent linkages, with the adenine and thymine rich regions in the minor groove of DNA, significantly altering the chromatin structure [Exp. Cell Res. (1973), 81,474-477; Biochemistry (1990), 29.9029-9039] The dyes Hoechst 33342 and Hoechst 33258 are frequently used in cytometry to stain chromosomes tnsitu. [J. Histochem Cytochem(1985)>33,333,338]. These two bis-benzimidazole compounds become brightly fluorescent when they bind to DNA for a long time, it has been known that Hoechst 332S8 binds specifically to AT-rich sequencer in DNA [Biochim. Biophys. Acta 1987, 949, 158-168; J. Mol. Biol. 198? 197, 257. Biochemistry 1991, 30, 182 189 Biochemistry 1991, 30, 10294-10306 BMBO J 1992, 11, 225-232]. A variety of NMR and X-ray crystal structures of Hoechst 33258 bound to different oligonucleotide duplexes have been published [Biochemistry (1989) 28, 7849-7859; Nucleic Acids R»e (1990) 18, 3753-3762; J. Chem. Sot Chem Common. (1991) 1770; Eur. J. Biochem (1993) 211, 437-447; Biochemistrv (1991) 30, 11377-11388]. Collectively, these structural studies reveal that the drue tits snugly into the minor groove of the double helix, covering a run of four contiguous AT base pairs. The Hoechst 33258-DNA interaction appear to be stabilized by several H-bonding and van der Waali contacts [Structure 1, 177] but in fact these molecular forces are believed to contribute to overall binding affinity [J. Mol. Biol (1997) 271 244-257], The hydrophobic transfer of hgand from solution on to its DNA binding Kites is more likely to represent the main driving force for the complex formation [J Mol B»ol (1997) 271, 244-257]. Administration of these compounds prior to irradiation afford protection against the formation of pnmary lesions in the aqueous solutions of DNA m well as in the intact cell nucleus. These DNA Uganda have also been observed to reduce the radiation induced cytogenetic damage
and cell death in cell cultures, as wall a& in whole body irradiated animals [Ind J Lxp Biol (1998) 36, 375-384; Br J Cancet (1989) 60 715-721]. However, post-irradiation treatment of cells with these ligandfe has. been observed to enhance cell death in vitro [IndJ.Exp.Biol(1998)36,37S-384], Fr*e radical scavenging and quenching o DNA radicals appear to be the mechanisms responsible for protection by Hoechst compounds administered prior to irradiation {int J Radiation Oncol. (1992) 23, 579-s86, Radioprotection (1997) 32,01-89], but its role in enhancing the radiation- induced cell death when administered after irradiation ta riot clearly understood The limitations of these minor groove binding Uganda a being mutagenic, clatogenic and cytotoxic because of the DNA lesions caused on account of topoisomerase I inhibition, gene expression alteration and repair inhibition prevent them from being used in humans Therefore., the development of DNA binding Uganda (Minor Groove Binding Ligands particularly) mat afford radioprotective effect without significant mutagemcity and cytotoxic effects can play a significant role in biological radiation protection.
OBJECTS OF THE INVENTION
An object of the invention is to synthesize benzimidazoles and its derivatives.
Further object of this invention is to avnthesize benzimidazoles having pharmacological activity.
Yet another object of this invention is to synthesize benzimidazoles DNA ligands which can afford radioprotective effect without significant mutagenicity and cytotoxic effects
DETAILED DESCRIPTION OF THE INVENTION
According to this invention there is provided a process for the synthesis of bisbenzimidazoles and its derivations comprising:
i) reacting 5 chloroaniline with zinc dust and acetic anhydride to produce 5 chloroacetanilide;
ii) reacting 5 chloroacetanilide with HNOa to produce 2-nitro-5-chloroacetanilide;
iii) boiling sodium methoxide and 2-nitro-5-chloroacetanilide under reflux for 3 hours to prepare 2-nitro-5-chloroaniline;
iv) heating 2-nitro-5-chloroaniline, methyl piperazine, anhydrous KaCOa and Dimethyl formamide at 100-120°C produce a mixture which is cooled by pouring ice and is filtered to obtain 5-(4'-methylpiperazin-1 '-yl)-2-nitroaniline;
v) treating 5-(4'-methylpiperazin-l'-yl)-2-nitroaniline with Pd/C to produce 2-amino-4-(4'-methylpiperazin-l'-yl) aniline;
vi) refluxing a mixture of 2-amino-4-(4'-methylpiperazin-l'-yl) aniline and ethyl-4-amino-3-nitrobenzenecarboximidate hydrochloride in presence of ethanol/glacial acetic acid to produce 4-[5'-(4"-methylpiperazin-l"-yl) benzimidazol-2'-yl)-2-nitroaniline;
vii) treating a solution of 4-[5'-(4"-methylpiperazin-l"-yl) benzimidazol-2'-yl)-2-nitroaniline with palladium on carbon to yield 2-amino-4-[5'-(4"-Methylpiperazin-l"-yl)benzimidazol-2'-yl]aniline;
viii) heating 2-amino-4-[5'-(4"-Methylpiperazin-l"-yl) benzimidazol-2'-yl] aniline and 3-4-dimethoxy benzaldehyde using nitrobenzene as a solvent at 110-150°C to produce (DMA) i.e. 5-(4-methylpiperazin-1-yl) -2- [2'- (3,4- dimethoxyphenyl) -5' benzimidazolyl] benzimidazole;
ix) heating 2-amino-4-[5'-(4"-Methylpiperazin-l"-yl) benzimidazol-2'-
yl] aniline and 5-Formyl-[3-methoxy-4-hydroxy benzimidazole]
using nitrobenzene at 110°C to 150°C in presence of argon to
produce (TBZ) i.e. 5- (4-methylpiperazine-l-yl) -2- [2'{2"-
(4- hydroxy- Smethoxyphenyl) 5" benzimidazolyl} -5'-benzimidazolyl] benzimidazole.
methlypiperazine-1-yl) 2 {2 {1" (4-hydroxy-3methoxyphenyl)5>'benzimidazolyl) 5'- benzimidazolyl] benzunidazole
5-Cyano-2 [3-methoxy-4-hydroxy benzimidazole] 5 -yl is produced by reacting M Diamino benzonitrile and 3-mrthoxy-4-hydroxy benzaldehyde. 5-Cyano-2 [ 3-methoxy-4-hydroxy benzimidazole. 5-yl is then treated with Raney oicke. in presence of formic acid to produce s Fonnyl-[3-inethoxy-4-hydroxy benzimidazole j
The complete reaction is shown in the accompanying reaction scheme.
The pharmacological activities ot two benzimidazoles having bisubstituted phenyl ring are described. The relative pharmacological activity of
5-(4-methylpiperasin-1'yl)-2 [2 -(3.4-dimethoxyphenyl)-5'-benzimidazolyl
bennnndacol (DMA) and 5-(4-methylpiperasin-1'yl)-2-[2'{2r (4-
hydroxyJmethoxyphenjl) 5"beuanidazolyl} -5'-benxfandacolyl] benzimidazole (TBZ) was compared to mat of Hoeciist 33342 The two compounds on the basi^ ot earlier observation that Hoechst 33342 ^ more cytotoxu: man Hoechst 33258 as the para phenolic group of Hoechst 33258 it- being replaced by emoxy group in Hoechst 33342 Keeping above observations in mind we have introduced two methoxy group in the biabenziniidaole and one methoxy mid one hydroxyl group in trisbenzinu'dazoie The synthesis of bisbenzimidazole was earned out basically as described by Kelly et al (1994) using 2-Amino-4-[5'-(4"-Methylpiperaan-l -yl)benzimidazol-2'-yl]aniline and M-dimethoxy benzaldehyde in 30% yiaiu Ilu» methodology is different from the method described by Lown et al. where tht-y have condensed 2-arylbenzimidazole witft o-arylenediamine . The terbenzimidazolc was prepared using a methodology described bv Edmond J. La Voie (1995) ustti^ tin equimolar mixture of 2-Amino-4-[5'-(4 Methylpiperazin-r'-yl)benzimidazol-2 yljamline and 5-Formyl-[3-memoxy-4-hydroxy benzimidazole] in nitrobenzene at 14t> 1 S0°i for 36 h in 25% yield. In this reaction the initially formed schiff base undergoes oudative cychzation by nitrobenzene to give
terbenzimidazole. However, we introduced two important modifications in bisbenzimidazole and terbenzimidazole This ts the first report of synthesis ot terbenzimidazole having bisubstitutiou on the phenyl ring. In the bisbenzimidazole the two raethoxy groups are introduced which are electron donating whereas tn the terbenzimidazole one methoxy group was replaced by hydroxyi group to observe the stability of DNA-drug complex using hydroxyi group capable of hydrogen bonding.
Both of the above mentioned compounds are non-cytotoxic even at l00µM concentration even upto 72 hours attei treatment in human glioma cell line BMG-1 cell survival assay showed that 5-(4-methylpipe.razin-l-yl)-2-[2'-(3,4-dimethoxyphenyl)-5 benzimidazolyl] benzimidazole (DMA) has 23% cell smrvival at lOOjiM concentration whereas5-(4-memylpiperazin-l-yl)-2-[ 2{ 2"-(4-hydroxy-3memoxyphenyl) 5"benzimidazolyl}-5'-benzimidazolyl| benzimidazole (TBZ) at lower concentration , e , KtytM has shown increase in growth whereas at l00µM showed 92% cell survival These results are further supported by growth kinetics
Under the same conditions, the ceils were irradiated with 2Gy, 5Gy and l0Gy of radiation and physico-chemical studies like UV-Vis spectroscopy, fluorescence spectroscopy and thermal denaturatiou studies were done. Cytotoxicity, cell survival assays were performed in vivo in human brain glioma cell line (BMG-1). The studies revealed that the bisubstimted minor groove binding analogues of Hoechst 33258 showed high degree of protection against high doses of radiation also.
Objective of the Invention
The cytotoxic effect of DMA and TB/ on exponentially growing tumor cells BMG-1 was studied as a function of time. Cells were treated for 1 h with the ligands and allowed to grow for 24, 48 and 72 hours. The MIT assay was performed at the specified time point* It was observed that Hoechst 33342 was highly cytotoxic at l0µM concentration whereas DMA and TBZ do not show any cvtotoxicity even at 100µM concentration. Inhere deemed to be no effect on the metabolic atatus of the treated cells. However, strikingly
the metabolic activity of the treated cells seemed to be enhanced as compared to the untreated control cells. To further support our results we have performed cell survival assay at 0.lµM, l0uM and l00uM concentration of drug having control as untreated cell These experiments were done thrice in six sets Exponentially growing cells were used in these experiments and cell survival was studied using Macrocolony assay. The plating efficiency was nearly 77%. The parent compound Hoechst 33342 showed a significant effect on cell survival. At low concentrations, Hoechst 33342 do not seem to have considerable effect on cell survival whereas at 10µM concentration the surviving fraction reached remained 45% and at l00µM only 10% of cells survived DMA at low concentration (0.1,1µM) did not show any effect on survival but at l0µM mere is a slight (3%) decrease in surviving fraction and at l00µM, 73% of the cells survived. TBZ had a significantly different behavior than the two ligands. At low concentrations (upto l0µM) , TBZ seemed to have a slight stimulatory effect on growth whereas at l00µM showed a little (9%) decrease in surviving fraction The effect of DMA and TBZ on proliferation of exponentially growing cells at 100µM concentration was also studied. The growth kinetics of the treated cells did not differ from the untreated cells. The increase in ceil number as a function of time for me control as well as the treated cells remained same The radioprotective effect of the newly synthesized ligands were studied spectroscopically as well as in human bran glioma cell line BMG-1. The spectroscopic studies done included UV-Vis sepctroacopy. Fluorescence spectroacopy, Anisotropy measurements and Lifetime measurements and Thermal denaturation studies Calf thymuB DNA was chosen as the DNA material and ligands were incubated with the DNA and irradiated at different doses of radiation The irradiated samples did not show any difference in the measurements as compared to the control DNA sample. The degree of DNA helix stabilization is considerably increased in the presence of the radiation and thus no DNA strand breaks were detected. At the cellular level also, the ligands were incubated with the cells and celt survival assay was performed to study the radioprotective effect of the new Uganda in comparison to the parent molecule Hoechst

33342. The new ligands showed rugh degree of radioprotection even at tower concentrations of the liganda.
Hie property of being non cytotoxic coupled with the radioprotective effect of the new ligands make them potential candidate for protection against radiation.
Examples
Example 1:
l-Nttro-5-ciyorauriIinc : A solution oi metallic aodium(0.012 g, 0.52mmol) in 44m) of
absolute methanol was added to 2 aitro-5 chloro acetanilide (4.8 g, 23mmol) the
solution was boiled under reflux for 8h and then the solvent was evaporated to obtain the
desired yellow crystalline product in 98% yield m. p. 126-128° C
Example! :
5-(4'-methylpiperaxin-1'yl)-2-nitroaniline: A stirred mixture of 2-nitro 5
chloroaniline(3.4g, 19.761181101), 1 -methylpiperazme (4.4ml, 52mmol), anhydrous K2CO3
and dry DMF (41ml) was protected with a CaCl2 drying tube and heated at 120°C for
20h. Upon cooling the mixture was poured into 300ml ice cold water and filtration of the
resulting suspension afforded a yellow solid Recrystallized using CCl4 to give lustrous
bright yellow plates to get 4.23 7g (100% yield) of the title compound.
m. p. : 152° C IR : 1HNMR : 5 2 27(s,3H , NCH3), 2.47( m,4H,H 3',5'), 6 (d, J 8Hz,
1H, H6), 6.37 (dd, J10, 3Hz, 1H, H4), o 89(sv 2H, NH2)
Example 3 :
2-Amino-4-(4'-methylpiperasin-1'yl) amiline : A solution of 5-(4'-methylpiperazm
l'yl)-2-nitroaniline (2.275g, 9.63mmoi ) in methanol/ethyl acetate (20:80, 98 ml) was
treated with 5% Pd/C ( 500 mg) and the mixture was hydrogenated at room temperature and atmospheric pressure. After the reaction is complete, the solution is color!rsis Filtration (celite) and concentration oi the filtrate without delay afforded the pale yellow colored diamine (4a) in 100%yield Example 4 :
4-Ammo-3-iritrobeiutoiiitrile: 4-cyano 2 mtroacetanilide (3gf15mmol) was heated under reflux in 10% HjSO* for 30 mia Upon cooling the resulting precipitate was filtered off and dried. Recrystallization of mis material (methanol/water) gave yellow crystals ol 4-amino-3-nitro benzonitrile in 95% yield rn. p. 160°C Example 5:
EthyM-Ainmo-3-iiitrobeiixenec*rbo;ninM!att hydrochloride : 4 -ammo -3-nitrobenzonitrile (2g ,12.5mmol) wa» suspended in dry ethanol (200ml at lOmg/ml concentration)and cooled in an ice/water bath Anhydrous HCl gas was bubbled rapidly through the mixture for 30-45 mm During mis time, dissolution followed by rapid precipitation was observed. The vessel was fitted with a Cad? drying tube and the thick suspension was stirred overnight The ethanol was removed by rotary evaporation and the pasty residue was triturated with dry dtethyl ether Filtration, followed by drying under reduced pressure afforded the title tmino ether hydrochloride as a hygroscopic bright yellow powder in 80 % yield m. p 233°<: ib. cm nmr t j och2ch3 j7hz. d h6 h2> Example 6 :
4-[5'-(4"-methylpiperazin-l"-7l)benxiniidazol-2'-yl]-2-nttroanfline : A mixture of freshly prepared 2-Amino-4-(4'-methylpiperaztn-r-yl) aniline (2.01 g, 9.75mmol) and Ethyl-4-Amino-3-mtrobenzenecarboxunidate hydrochloride (1.98 g, 9 5mmol) in dry ethanol/ glacial acetic acid (2:1,69 nil) was maintained under nitrogen and heated at reflux for 4 h. The mixture was cooled and then concentrated to afford an orange pasty residue. This material was dissolved m water and addition of cone. Ammonia solution resulted in precipitation of a solid, thr suspension was allowed to stand overnight the brick red solid was coiiected by filtration and washed thoroughly with water before being dissolved in acetic acid/ methanol (75 92 5), 50 ml. The deep red solution was filtered and men made alkaline with cone. Ammonia solution (c. 20ml) .A fine orange precipitate formed immediately and the suspension was allowed to stand for several hours betbre filtration, The solid thus retained was washed thoroughly with water and then with acetone. Drying under reduced pressure afforded an orange solid in 74% yield m. p 184-186°C,
IR : 3494, 3365, 2939, 2808, 1639 1510. 1247, 796 cm "-1.1H NMR : δ 2.99(s,3H, NCH3), 3.16 (m,2H), 3.34 (m,2H), 3 6/ (m,2H), 3.94 (m,2H), 7.24 (dJ9Hz,lH), 7 25(d, J2Hz, 1H;), 7.36 (dd, J9Hz, 1H), 7.98 { dd, J 9 2 Hz, 1H), 8.95 (d, J 2Hz, 1H) Example 7 :
2-Amino-4-[5'-(4"-Methyhpiperaxm-r yl)benzimidazol-2 -}l]anitine :
A solution of 4-[5'-(4"-methylpiperazm-1"-yl)benziniidazol-2'-yl]-2-nitroaniline (1 04 g 30mmol) in ethyl acetate/methanoi (80 ml of 2:1 mixture) was treated with 5% palladium on carbon (250 mg) and hydrogenated at room temperature and atmospheric
pressure. When hydrogen uptake has ceased the solution was filtered (celite) and concentrated without delay to afford the- orange-brown colored diamine. Example 8 :
3-Cyano-2 [3 mtthoxy-H hydroxy faemfanldazole] 5' yl : A solution of 1.224 g (9 2 mmol) of previously obtained 3,4-Dianuno benzonitrile , 1.4 g (9.2mmol) of 3-methoxy-4-hydroxy benzaldehyde in nitrobenzene are taken in a. three necked round bottom flask under nitrogen and heated at 140° C Fhe reaction mature is heated for 18 hours with stirring, nitrobenzene is then removed under reduced pressure to obtain the brown colored crude product (14). The final product is then obtained through silica gel (60 i 20 mesh size) column chromatography using EtGAc / MeOH as eluent. m.p 226-230° ( IR 3425.3 (O-H), 3263.3 (-NH), 2221 8 -CN), 1278.7 (C-O-C) cm1; }H NMR 5 13 5 (B, 1H, -NH), 9.52 ( B, 1H, -OH),7.85 (d 1H. C7). 7,82 (d, IH, C6), 7.72 (s, 1H, C3') 763 (d,lH,C5')> 6.93 (d, 1H, C6) 3.90 (8,34, -OCH3) Example 9 :
5-Formyl-[3-metlioxy-4-hydroxy bennmidaxole] : To a solution of 1 g (3 77mmol) 5 Cyano-2 [3-methoxy-4-hydroxy benzinudazole] 5'-yl in 60ml formic acid and 20 ml of water, Raney nickel (3.96 g) was added The reaction mixture was heated at 95° C for 6 hours. The hot mixture was filtered (ceiite) and the reaction flask and the celite bed were rinsed with water. The aqueous solution was concentrated to dryness. To this residue, water is added to obtain a white precipitate, The pH of this suspension was adjusted to 9 by the dropwise addition of 2 N Na OH The product was obtained by extraction with ethyl acetate. The ethyl acetate extraci was dried (Na2SO4) and concentrated in vacuo to give the yellow colored compound in 30% yield, m. p. 258 -260°C ; IR 3435.6 ( H) ,3194,55 (NH) , 1675.6 (CHO), 1594 1506 1441.6, 1280.7cm ' ; 'HNMR 6 13 7 (b. 1H, -NH) , 10.2 (S,1H, -CHO) ,9.5 (s 1H, -OH). 8.25 (d,1H, C6), 7.78 (s.lH, C4). 75 (d,1H C7), 7.7 (s, 1H, C3'), 7.65 (d,lR C5'), 6 92 (d,1H, C6'), 3.92 (s,3H, -OCH3) Example 10 : 5 (4-methylplperazin-1yl)-2-[2' (3,4 dtmethoxyphenyl) 5' benxlmldazolyl]
benazinmidazole : A solution of freshly prepared 2 Ammo-4 [5'-(4"-Methylpiperazin 1
yl)benzimidazol-2'-yl]aniline (1.18g,3 67mmol) and 3,4-diniemoxy benzaldehyde ( 0 6lg ,3.67mmol) in nitrobenzene (110 ml) is heated at 140-150° C for 24 h. The solvent is then removed under reduced pressure to give the final crude product as a brown colored sohd The product is purified by column chromatography on BUCHI 688 Liquid (MPLC) Pump using silica gel (70-230 mesh size) and EtOAc/ MeOH as elueot to give a yellow colored compound and characterized by apectroacopie techniques .m. p. 220°C Yield 30%; IR : 3556, 2922, 1629 08, 1417 1371, 1296, 1022, 810cm-'; 1H NMR (DMSO-d6) δ 2.24 (s,3H,CH3), 2 46 (t,4H.CH2,J=4Hz): 3.5 (t,4H,CH3, J=4H2) 1 86 (s,3H,OCH3), 6.77 (d,lH,Ar-nJ=8Hz) ' 13 (dv2RAr-H, J= 9Hz), 7.64 (d,lIiAr-HJ=8.5Hz ), 7.78 (d,lH,Ar-HJ=8 Hz,, 8.03 (mJH,Ar-H),8.17(d>2H,Ar-h) J=9Hz), S ? (sJH^Ar-H), 13.0 (bs,2H>NH) .D2O exchange resulted in the disappearance of the peak at
613.0.;EIMS : 467 (M+),425,411. 2ol 235 220,194, 118, 91, 55,44 Example 11:
5 (4 methylplperaznvl yf) 2 [2'{2 ,4 hydroxy-3metltoxyphenyI)5'benzimIdazolyl} -5'-benzimidazolyl] benztanidazole . A aolution of freshly prepared 2-Amino-4-[5' (4 -Methylpiperazui-l"-yl)beazimidazol-2 -yljanilme (218 mg, 0.82mmol) and 5-Formyl-! ?-methoxy-4-hydroxy benzimidazolej (182 tng .U68mmol) in nitrobenzene IB taken under argon in a round bottom flask and heated at 140°C for 20 hours. Nitrobenzene is removed under reduced pressure and the resulting aoltd is purified by column chromatography (EtOAc/ MeOH) on BUCHI 688 Liquid (MPLC) Pump using silica gel (70-230 mesh size) to obtain the final product (25% vield) as a brown colored solid. m.p. >290° 13,4-13.55 ( b,3H), 9.5 (s, 1H), 8.4 The cytotoxicity was deternnneo using the MTT microtiter plate tetrazohuni cytotoxicity assay (MTA). The human brain malignant glioma cell line BMG-1 was used The cytotoxicity assay was performed USing 96 well microtiter plates. 3000 cells/ well
ere plated and treatment of the ligands was done 24 hours post plating. For determination of IC50 , cells were exposed continuously with varying concentration of drug and MTT assays were performed at the end of fourth day. To the control and treated cells , 20 pi of 5mg/ml MTT in PBS wan added, incubated for 2 hours at 37°C and then the medium was removed 150 µl at DMSO was added and the plate was read at 540 nrn with reference at 630 ran . Macrocolony An ay
Cells were washed with HBSS and harvested using 0.05% trypsin Depending on die treatments, 200-1200 cells were plated in 90-mm Petri dishes and incubated at 3 7°C in a 5% CO2 humidified atmosphere tor 8-10 days. Colonies were fixed inmethanol and stained with 1% crystal violet Colonies containing more than 50 cells were counted as shown in Table 1&2.
Proliferation Kinetics BMG-1 -ells were seeded at 7000-8000 cells/ cm and
their proliferation kinetics was measured at 24-h intervals by trypsinizinig and counting total cells per flask using a hemocytomeiet
Table 1
Thermal Denaturation studies of figand-DNA complex in presence and absence of radiation
(Table Removed)
Table 2
Cell Survival Assay in BMG-1 cells with and without irradiation

(Table Removed)
Experiment with Normal human embryonic kidney ceil fine (Hek cell fine):
To demonstrate the effect oi DMA and TBZ as radioprotectors in comparison to Hoechst 33342, we also chose a nonnai (untrangformed) human embryonic kidney cell line. Though the two cell lines, Hek and BMG-1 ceil line are not radiobiologically different, it was still reasonable to demonstrate the invitro effect of these ligands on different cell lines.
The results with the two cell ltnea are not significantly different, but it can be concluded that both DMA and TBZ are better radioprotectors in comparison to Hoachst 33342.
Below presented are me results of the growth kinetics and Macrocolony assay performed with the two cell lines.
Procedure:
Macrocolony A»ay (CeD Survival Assay)
Both cell lines in triplicate were washed with HBSS, plated and grown for tour days before harvesting using 0.05% trypsm. Depending on the treatments, 200 to 1200 cells were plated in 90 mm petri dishes and incubated at 37°C in 5% CO2 humidified atmosphere for 8 to 10 days. Colonies were fixed with methanol and stained with 1 % crystal violet. Colonies containing more than 50 cells were counted. Proliferation Kinetics
Bom the cell lines in triplicates were seeded at 7000 to 8000 cella/ cm2, and their proliferation kinetics was studied at 24 h intervals following trypsinization and counting total cells per flask using a haemocytometer Irradiation Procedures:
Exponentially growing cells 24 h after plating were irradiated af room temperature in growth medium with a to-60 source (Gamma cell, AECL, Canada) m a dose rate of 0.5 tol.2 Gy/mia Concentration of lOµM drug was added 1h before irradiation. Following this procedure, cells were grown for various time intervals to study growth kinetics and plated for Macrocolony assay as shown in the following Tables 3 & 4 and figure 1 & 2

Table 3
CeD Survival Assay in BMG 1 cells with 5 Gy irradiation


(Table Removed)

Table 4
Cefl Survival Assay in Hek cells with 5Gy irradiationt
(Table Removed)






WE CLAIM:
1. A process for the synthesis of bisbenzimidazoles and its derivations comprising:
i) reacting 5 chloroaniline with zinc dust and acetic anhydride to produce 5 chloroacetanilide;
ii) reacting 5 chloroacetanilide with HNO3 to produce 2-nitro-5-chloroacetanilide;
iii) boiling sodium methoxide and 2-nitro-5-chloroacetanilide under reflux for 3 hours to prepare 2-nitro-5-chloroaniline;
iv) heating 2-nitro-5-chloroaniline, methyl piperazine, anhydrous
KaCO3 and Dimethyl formamide at 100-120°C produce a mixture which is cooled by pouring ice and is filtered to obtain 5-(4 -methylpiperazin-1 '-yl)-2-nitroaniline;
v) treating 5-(4'-methylpiperazin-l'-yl)-2-nitroaniline with Pd/C to produce 2-amino-4-(4'-methylpiperazin-I'-yl) aniline;
vi) refluxing a mixture of 2-amino-4-(4'-methylpiperazin-I'-yl) aniline and ethyl-4-amino-3-nitrobenzenecarboximidate hydrochloride in presence of ethanol/glacial acetic acid to produce 4-[5'-(4"-methylpiperazin-l"-yl) benzimidazol-2'-yl)-2-nitroaniline;
vii) treating a solution of 4-[5'-(4"-methylpiperazin-l"-yl) benzimidazol-2'-yl)-2-nitroaniline with palladium on carbon to yield 2-amino-4-[5'-(4"-Methylpiperazin-l"-yl)benzimidazol-2'-yl]aniline;
viii) heating 2-amino-4-[5'-(4"-Methylpiperazin-l"-yl) benzimidazol-2'-yl] aniline and 3-4-dimethoxy benzaldehyde using nitrobenzene as a solvent at 110-150°C to produce (DMA) i.e. 5-(4-methylpiperazin-1-yl) -2- [2'- (3,4- dimethoxyphenyl) -5' benzimidazolyl) benzimidazole;
ix) heating 2-arnino-4-[v5'-(4"-Methylpiperazin-l"-yl) benzimidazol-2'-
yl] aniline and 5-Formyl-[3-methoxy-4-hydroxy benzimidazole]
using nitrobenzene at 110°C to 150°C in presence of argon to
produce (TBZ) i.e. 5- (4-methylpiperazine-l-yl) -2- [2']2"-
(4- hydroxy 3methoxyphenyl) 5" benzimidazolyl} -5' benzimidazolyl] benzimidazole.
2. A process for the synthesis of bisbenzimidazoles and its derivations substantially as herein described and illustrated.

Documents:

32-del-2003-abstract.pdf

32-del-2003-claims.pdf

32-del-2003-complete specification (granted).pdf

32-del-2003-correspondence-others.pdf

32-del-2003-correspondence-po.pdf

32-del-2003-description (complete).pdf

32-del-2003-form-1.pdf

32-del-2003-form-19.pdf

32-del-2003-form-2.pdf

32-del-2003-form-3.pdf

32-del-2003-form-5.pdf

32-del-2003-pa.pdf


Patent Number 241650
Indian Patent Application Number 32/DEL/2003
PG Journal Number 30/2010
Publication Date 23-Jul-2010
Grant Date 17-Jul-2010
Date of Filing 09-Jan-2003
Name of Patentee UNIVERSITY OF DELHI
Applicant Address CENTER FOR BIOMEDICAL RESEARCH, UNIVERSITY OF DELHI, DELHI-110 007
Inventors:
# Inventor's Name Inventor's Address
1 TANDON VIBHA CENTER FOR BIOMEDICAL RESEARCH, UNIVERSITY OF DELHI, DELHI-110 007
2 - CENTER FOR BIOMEDICAL RESEARCH, UNIVERSITY OF DELHI, DELHI-110 007
3 JAIN AKASH CENTER FOR BIOMEDICAL RESEARCH, UNIVERSITY OF DELHI, DELHI-110 007
4 TAWAR URMILA CENTER FOR BIOMEDICAL RESEARCH, UNIVERSITY OF DELHI, DELHI-110 007
5 CHANDRA RAMESH CENTER FOR BIOMEDICAL RESEARCH, UNIVERSITY OF DELHI, DELHI-110 007
6 DWARAKANATH BILIKERE SRINIVASRAO CENTER FOR BIOMEDICAL RESEARCH, UNIVERSITY OF DELHI, DELHI-110 007
7 CHAUDHURY NABOKUMAR CENTER FOR BIOMEDICAL RESEARCH, UNIVERSITY OF DELHI, DELHI-110 007
PCT International Classification Number A61K 031/4184
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA